Project Summary. With over 55,000 individual structures reported in the literature, isoprenoid compounds constitute the most chemically diverse collection of compounds found in nature. Members of this group are essential to support life in all organisms except for a few parasitic bacteria with very small genomes. Isoprenoid molecules function as hormones, as structural components in membranes, as lipophilic moieties that anchor cofactors and proteins to membranes, in vision, as mating pheromones, and as defensive agents, to name a few. Isoprenoid molecules are used to treat cancer and regulation of isoprenoid metabolism is the basis for highly successful treatments of coronary heart disease. The early building steps in the isoprenoid biosynthetic pathway, where the basic carbon skeletons are constructed, are condensation reactions between electron-rich nucleophilic acceptors and electrophilic allylic diphosphate esters. The prenyltransferases that catalyze these reactions belong to a large structurally diverse family of enzymes that mediate alkylation of nucleophilic carbon-carbon double bonds, aromatic rings, amino groups, hydroxyl groups, and thiol groups. The long term goal of this project is to determine how prenyltransferases catalyze reactions, to study how changes in the structures of prenyltransferases lead to new activities, and to use prenyltransferases to develop new technologies based on introduction of unnatural amino acids into proteins. To facilitate our work, we are developing a collection of genetically engineered strains of Escherichia coli that permit us to rapidly produce and purify multimilligram quantities of prenyltransferases for study in our laboratory and to provide proteins to collaborators for X-ray studies. We are also synthesizing substrates, putative products, and inhibitors, including molecules with stable or radioactive isotopes, for our mechanistic studies. These compounds are typically not available commercially and are often highly reactive. Our work has led to several new practical procedures for their synthesis and to development of compounds useful for assaying prenyltransferase activity. Protein microarrays are increasingly being used for high-throughput screening in drug discovery and clinical analysis. We are developing reagents and protocols for regio- and chemoselective attachment of proteins to silica and gold surfaces that permit us to attach natively folded proteins in a manner that preserves their function. The general approach involves posttranslational modification of a cysteine residue in the protein to create an unnatural amino acid bearing a bioorthogonal functional group, which can then be used to covalently anchor the protein to another molecule or a suitably derivatized surface. This approach can also be used to regio- and chemoselectively attach proteins to other molecules, and offers a new approach for synthesis of clinically useful protein-polyethylene glycol conjugates.